An immunochromatographic strip immunoassay has become common as a convenient in vitro diagnostic kit or a portable diagnostic device for the detection of a target substance (such as antigens) in a sample solution using the specific reactivity of antibodies.
Lately, a convenient immunochromatographic testing tool that detects the presence or absence of an infection caused by pathogens such as influenza viruses and bacteria has attracted interest, and there has been ongoing research and development of such tools.
Hemolytic streptococcus (hereinafter, also referred to as “streptococcus”) infections are diagnosed by a test that uses group-specific polysaccharides as antigens. Known methods of polysaccharide extraction use, for example, enzymes, phages, hydrochloric acid, and hypochlorous acid. The extraction method that uses nitrous acid is most common.
The extraction method using nitrous acid has a number of merits, including high extraction efficiency for polysaccharides, and the low price and the easy handling of nitrous acid. A drawback, however, is that the nitrous acid itself is an unstable, easily decomposable compound, and needs to be prepared fresh by mixing sodium nitrite and an organic acid such as acetic acid before extraction.
Such preparation of nitrous acid in each case is highly laborious for physicians and laboratory technicians when diagnosis is made on a regular basis. Further, because the process involves a mixing step, there is a possibility that the method may produce an inaccurate and unsafe diagnosis due to errors in mixing reagents. Another drawback is the poor assay repeatability due to the extraction time.
In order to overcome these drawbacks, there has been research and development of convenient testing tools that will simplify the extraction of polysaccharide antigens from hemolytic streptococci.
For example, patent document 1 proposes a simplified extraction method for the extraction of polysaccharide antigens from an organism (particularly, group A or B streptococcus) using a kit that combines a) a first absorbent material that is dried after being impregnated with a premeasured amount of a nitrite salt, b) a second absorbent material that is dried after being impregnated with a premeasured amount of a neutralizing base and a buffer, and c) a premeasured amount of an aqueous solution of an acid (see patent document 1).
A diagnosis of streptococcus infection is also a laborious process that requires first extracting a polysaccharide antigen, and then detecting the antigen by contacting the resultant solution with an immunoassay device. This has created a need for developing a method or a kit that allows for assaying an organism in a sample with ease with the step of extracting a polysaccharide antigen, and the step of measuring a marker.
For example, patent document 2 discloses a device and method for the detection of carbohydrate antigens that are characteristic of microbial/bacterial organisms, such as the family Streptococcacae. The lateral flow assay device provided in this publication includes a substrate having a) a sample receiving zone, b) an extraction zone (extraction reagent; an acid and a nitrite salt that are immobilized or absorbed and dried), c) neutralizing agent (neutralization buffer; TRIS), and d) a detection zone (capture/detection reagent) (see patent document 2).
A method and a kit for simultaneously assaying a plurality of organisms including streptococci are also developed. For example, patent document 3 describes a method and a kit for the measurement of a plurality of different biological species in a sample in which a first organism is a gram-positive bacterium, for example, a group A, B, F, or G streptococcus, and enterococcus bacteria, and a second organism is a virus, or a gram-negative bacterium.
The invention disclosed in patent document 3 is intended to enable a simultaneous detection of more than one analyte using a kit that includes, in one or more containers, a) nitrous acid, or an acid or a nitrite salt in a dry form, b) a surfactant, c) a first binding reagent that binds a first marker from a first organism, and d) a second binding reagent that binds a second marker from a second organism. This enables doubling the test efficiency, and relieves the pain caused in a patient by the test. The method also allows therapeutic agents that are effective against a plurality of causal organism types to be administered in an appropriate combination.
The nitrite salt is combined with acids such as inorganic acids (e.g., hydrochloric acid, sulfuric acid), and organic acids (acetic acid, citric acid). The preferred acids are organic acids, more preferably acetic acid. Acetic acid is used in Examples (see patent document 3).
A streptococcus test typically uses group-specific polysaccharide antigens because Streptococcus have many serotypes. The extraction method using nitrous acid is a known method of extraction of polysaccharides from bacteria. However, being an unstable compound, nitrous acid needs to be prepared fresh by mixing sodium nitrite and an organic acid immediately before use.
This is problematic because the nitrous acid preparation adds to the test steps, and involves poor assay repeatability due to variation in the preparation technique, and the extraction time. The present inventors have successfully simplified the procedures, and improved the assay repeatability with an immunochromatographic kit having two sample pads separately impregnated with sodium nitrite and citric acid. A problem, however, is the poor extraction efficiency due to the non-specific color produced when an increased amount of citric acid is impregnated.
A streptococcus infection is a respiratory infection that is common year round, and Streptococcus species represent a group of known causative microorganisms of common cold in Europe and the United States, in addition to Japan. Easy and quick immunochromatographic diagnosis kits are used to assist diagnosis. (About 3 million tests are conducted per year in the market.)
Assay devices and methods for the detection of carbohydrate antigens that are characteristics of microbial/bacterial organisms such as the family Streptococcacae are already commercially available in the market. Known examples of currently available immunochromatography reagents include the QuickVue DipStick Strep A (DS Pharma Biomedical), and the StrepA Test Pack—Plus OBC (Sanwa Kagaku Kenkusho). Slide latex agglutination reagents, for example, the A Strept AD “Seiken” (Denka Seiken), are also known.
In commercially available immunochromatography reagents, a streptococcus concentration of at least 1×106 CFU/mL is typically required for the direct method, in order to produce a positive test result in testing an analyte. This is problematic because, when the streptococcus concentration is less than 1×106 CFU/mL, an analyte that should produce a positive test result will be tested negative. Another problem is that an immunochromatographic test agent with a labeled antibody conjugated to an insoluble carrier typically has lower sensitivity than EIA, and produces an unclear line for a positive test result. Further, the traditional reagents involve a false positive, which wrongly indicates a positive result despite the absence of a target substance (such as antigens) in a sample solution.
In order to solve these problems, methods are proposed that mix a sugar or a water-soluble polymer compound into a development solvent. For example, a membrane assay using antibody-conjugated colored latex particles is proposed that uses an immunoassay latex composition containing at least one aggregation preventing agent such as sugar group, for example, such as monosaccharides, oligosaccharides, and sugar alcohols and polyalcohols thereof in a latex composition, and a basic buffer with an added protein, and having a pH of 9.0 to 9.8. In this way, the method prevents natural aggregation of latex particles, and increase of specific gravity, viscosity, and osmotic pressure to enable a high-sensitive immunoassay (see patent document 4).
Glycohemoglobin (blood hemoglobin with sugar attached to it), particularly hemoglobin A1c (referred to as “HbA1c”) with the glycated valine residue at the N-terminal of the hemoglobin β chain is widely used as an index suited for the diagnosis of diabetes. With regard to a particle immunochromatography assay of these hemoglobins, a detection method is recently proposed that includes (A) treating a red blood cell-containing measurement sample with a surfactant to expose the N terminal of the hemoglobin β chain to protein surface, (B) contacting the resultant sample to a water-insoluble state of cyclic polysaccharides (for example, the cyclic polysaccharides are immobilized on membrane or the like by chemical bonding, and form a polymer by themselves in a state of being kneaded into a porous resin), and (C) contacting the sample to antibodies or the like that recognize the N terminal of the particle-labeled hemoglobin.
In this way, the method prevents the constituent cyclic oligosaccharide or polysaccharide molecules of cyclic polysaccharides from dissolving in water and becoming non-diffusive upon contact between cyclic polysaccharides and water, which is a cause of inaccurate measurement due to the antibodies forming aggregates, and failing to develop on a membrane (see patent document 5).
With regard to the simplified analyte test method based on membrane assay, an analyte sample filtration method is proposed that can prevent a false positive or clogging while maintaining sensitivity (see patent document 6).
However, with the immunochromatography method (also referred to as “particle immunochromatography method”) using antibodies conjugated to an insoluble carrier (metal particles, colored latex particles, etc.), aggregation of the insoluble carrier still occurs depending on the measurement sample, measurement environment, and measurement conditions. This may cause non-specific reactions, and the slow development rate remains a problem. There accordingly is a strong need for the pursuit of a test agent that does not cause aggregation of insoluble carriers, or non-specific reactions, and therefore has a fast development rate in a particle immunochromatography method performed with different measurement samples under different measurement environments or conditions.